Process for making beta 3 agonists and intermediates

ABSTRACT

The present invention is directed to a process for preparing a compound of formula I-11 through multiple-step reactions.

TECHNICAL FIELD

The present disclosure relates to a process for making beta 3 angonistsand intermediates, ketoreductase (KRED) biocatalysts and methods ofusing the biocatalysts.

REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM

The official copy of the Sequence Listing is submitted concurrently withthe specification as an ASCII formatted text file via EFS-Web, with afile name of MRL-BRE-00026-KRED.txt, a creation date of Oct. 24, 2011,and a size of 9,617 bytes. The Sequence Listing filed via EFS-Web ispart of the specification and is incorporated in its entirety byreference herein.

BACKGROUND OF THE INVENTION

This application is directed to an efficient and economical syntheticprocess for making a compound of formula I-11 which can be used as anintermediate compound for making beta 3 agonists.

SUMMARY OF THE INVENTION

This application is directed to a multiple-step synthetic process formaking a compound of formula I-11. In one embodiment, a KRED enzyme isused in the multiple-step process.

DESCRIPTION OF THE INVENTION

Described herein is a process of making compound I-11 from compound I-1through multiple step reactions:

wherein R¹ is selected from the group consisting of C₁₋₆alkyl, benzyland phenyl.

In one embodiment, the multiple-step reactions from compound I-1 tocompound I-11 comprise the following steps:

(a) reacting compound I-4:

with an acetonide protection reagent selected from the group consistingof 2,2-dimethoxy propane, 2,2-diethoxylpropane, 2-methoxypropene andacetone, to produce compound I-5:

(b) reducing compound I-5 with a reducing agent at a temperature of 0°C. to 40° C. to produce compound I-6:

(c) oxidizing compound I-6 with an oxidizing agent in the presence of asolvent and a catalyst to produce compound I-7:

(d) reacting compound I-7 with phosphate compound A-4:

to produce compound I-8:

(e) reducing compound I-8 in the presence of a catalyst to producecompound I-9:

(f) reacting compound I-9 with an acid to produce compound I-10:

and

(g) reducing compound I-10 in the presence of a catalyst to producecompound I-11;

wherein P¹ and P² are each independently selected from the groupconsisting of Ac, Bn, Boc, Bz, Cbz, DMPM, FMOC, Ns, Moz, and Ts; and

R¹ is selected from the group consisting of C₁₋₆alkyl, benzyl andphenyl.

In one embodiment, the solvent in step (c) above is selected from thegroup consisting of THF, MTBE, CH₂Cl₂, MeCN, toluene and a mixturecomprising two of the foregoing solvents. In another embodiment, theoxidizing agent is selected from the group consisting of NaOCl, NaClO₂,hydrogen peroxide, pyridine sulfur trioxide, PCC, and DCC. In anotherembodiment, the catalyst is TEMPO or a TEMPO analogue.

In one embodiment, the reaction between I-7 and A-4 in step (d) iscarried out at a temperature of about 20 to 40° C. In anotherembodiment, the reaction is carried out in the presence of a solventselected from the group consisting of THF, MTBE, CH₂Cl₂, MeCN, tolueneand a mixture comprising two of the foregoing solvents.

In one embodiment, the catalyst used in the reduction step (e) isselected from the group consisting of Pd, Raney Ni, Pt, PdCl₂, andPd(OH)₂. In another embodiment, the reduction is carried out in thepresence of hydrogen gas.

In one embodiment, the acid in step (f) is selected from the groupconsisting of HCl, HBr, TFA, MeSO₃H, TfOH, H₂SO₄, para-toluenesulfonicacid, and RSO₃H wherein R is alkyl, aryl or substituted aryl.

In one embodiment, the reduction of step (g) is carried out in thepresence of HMDS. In another embodiment, the catalyst used is selectedfrom the group consisting of Pt on alumina, Pd on alumina, Pd/C,Pd(OH)₂—C, Raney Ni, Rh/C, Rh/Al, Pt/C, Ru/C and PtO₂.

In one embodiment, the multiple-step reactions from compound I-1 tocompound I-11 further comprise reducing compound I-3:

in the presence of a KRED enzyme to produce compound I-4:

In one embodiment, the KRED enzyme is selected from the group consistingof a polypeptide of SEQ ID NO. 1 and a polypeptide of SEQ ID NO. 2.

In another embodiment, a cofactor recycling system is also present inaddition to a KRED enzyme. Suitable cofactor recycling systems include,but are not limited to, a polypeptide of SEQ ID NO. 3 and a polypeptideof SEQ ID NO. 4.

In one embodiment, a KRED enzyme of SEQ ID NO. 1 and a cofactorrecycling system of SEQ ID NO. 3 are present in the reduction from I-3to I-4.

In another embodiment, a KRED enzyme of SEQ ID NO. 2 and a cofactorrecycling system of SEQ ID NO. 4 are present in the reduction from I-3to I-4.

In one embodiment, a cofactor molecule which can donate an electron isalso present in addition to a KRED enzyme. In one embodiment, thecofactor is selected from the group consisting of NADH and NADPH.

In one embodiment, the multiple-step reactions from compound I-1 tocompound I-11 further comprise reacting compound I-1:

with benzoyl chloride and a protecting reagent to produce compound I-3:

wherein P¹ is a protecting group selected from the group consisting ofAc, Bn, Boc, Bz, Cbz, DMPM, FMOC, Moz, and Ts; and R¹ is C₁₋₆alkyl or anaryl.

In one embodiment, R¹ is methyl, ethyl, propyl or butyl. In anotherembodiment, R¹ is methyl, ethyl, or phenyl. In one embodiment, P¹ isBoc.

In one embodiment, the conversion from compound I-1 to compound I-3 iscarried out through compound I-2:

wherein R¹ is C₁₋₆alkyl or an aryl; and P¹ is selected from the groupconsisting of Ac, Bn, Boc, Bz, Cbz, DMPM, FMOC, Moz, and Ts.

In one embodiment, the conversion from I-1 to I-3 comprises:

wherein R¹ is C₁₋₆ alkyl or an aryl; and P¹ is selected from the groupconsisting of Ac, Bn, Boc, Bz, Cbz, DMPM, FMOC, Moz, and Ts. In oneembodiment, R¹ is methyl or ethyl and P¹ is Boc.

In one embodiment, step (a) or (b) above is carried out in the presenceof a base. Suitable bases include, but are not limited to, NaOH, KOH,Na₂CO₃, K₂CO₃, NaHCO₃, K₃PO₄, Et₃N, i-Pr₂Net, and pyridine. In oneembodiment, the base is NaHCO₃, Na₂CO₃ or Et₃N.

In one embodiment, the protecting reagent in step (b) is Boc₂O.

In one embodiment, the conversion from I-1 to I-3 is carried out in thepresence of a suitable catalyst. In one embodiment, the catalyst isDMAP.

In one embodiment, the conversion from I-1 to I-3 is carried out at atemperature of 0° C. to 60° C., or more specifically, 0° C. to 40° C.,or even more specifically, 20° C. to 30° C. In one embodiment, thetemperature is 20° C. to 30° C.

In one embodiment, the above conversion from I-1 to I-3 can be carriedout using similar synthetic processes as described in Novel N→C AcylMigration Reaction of Acyclic Imides: A Facile Method for α-Aminoketonesand β-Aminoalcohols, Hara, et. al., Tetrahedron Letter, Vol. 39, page5537 (1998).

In one embodiment, conversion of compound I-3 to compound I-4 is througha dynamic kinetic resolution (DKR) reduction in the presence of a KREDenzyme:

wherein R¹ is C₁₋₆alkyl or an aryl; and P¹ is a protecting groupselected from the group consisting of Ac, Bn, Boc, Bz, Cbz, DMPM, FMOC,Moz, and Ts. In one embodiment, R¹ is methyl or ethyl. In oneembodiment, P¹ is Boc.

In one embodiment, the enzymatic reduction of I-3 to I-4 is carried outin a solvent. Suitable solvents include, but are not limited to, IPA,DMSO, DMF, DMAc, and combinations thereof. In one embodiment, thesolvent is DMSO.

In one embodiment, suitable temperature for the DKR reduction from I-3to I-4 is 0°-60° C., or more specifically, from 10°-40° C., or even morespecifically, from 20°-35° C. In one embodiment, the temperature isabout 30° C.

In one embodiment, a KRED enzyme coupled with a cofactor recyclingsystem and an NADPH cofactor is used to reduce compound I-3 to obtaincompound I-4. Suitable reaction conditions for the KRED-catalyzedreduction of I-3 to I-4 are provided below and in the Examples.

KRED enzymes belonging to class EC 1.1.1.184 are useful for thesynthesis of optically active alcohols from the correspondingpro-stereoisomeric ketone substrates and by stereospecific reduction ofcorresponding racemic aldehyde and ketone substrates.

KRED enzymes typically convert a ketone or aldehyde substrate to thecorresponding alcohol product, but may also catalyze the reversereaction, oxidation of an alcohol substrate to the correspondingketone/aldehyde product. The reduction of ketones and aldehydes and theoxidation of alcohols by enzymes such as KRED typically require aco-factor, most commonly reduced nicotinamide adenine dinucleotide(NADH) or reduced nicotinamide adenine dinucleotide phosphate (NADPH),and nicotinamide adenine dinucleotide (NAD) or nicotinamide adeninedinucleotide phosphate (NADP+) for the oxidation reaction. NADH andNADPH serve as electron donors, while NAD and NADP serve as electronacceptors. KRED enzymes often can use either the phosphorylated or thenon-phosphorylated co-factor.

KRED enzymes can be used in place of chemical procedures for theconversion of different keto and aldehyde compounds to chiral alcoholproducts. These biocatalytic conversions can employ whole cellsexpressing the ketoreductase for biocatalytic ketone reductions, orpurified enzymes, particularly in those instances where presence ofmultiple ketoreductases in whole cells would adversely affect theenantiomeric purity and yield of the desired product. For in vitroapplications, a co-factor (NADH or NADPH) regenerating enzyme such asglucose dehydrogenase (GDH) and formate dehydrogenase typically is usedin conjunction with the ketoreductase.

Examples illustrating the use of naturally occurring or engineered KREDenzymes in biocatalytic processes to generate useful chemical compoundsinclude asymmetric reduction of 4-chloroacetoacetate esters (Zhou, J.Am. Chem. Soc. 1983 105:5925-5926; Santaniello, J. Chem. Res. (S)1984:132-133; U.S. Pat. No. 5,559,030; U.S. Pat. No. 5,700,670 and U.S.Pat. No. 5,891,685), reduction of dioxocarboxylic acids (e.g., U.S. Pat.No. 6,399,339), reduction of tert-butyl(S)-chloro-5-hydroxy-3-oxohexanoate (e.g., U.S. Pat. No. 6,645,746 andWO 01/40450), reduction pyrrolotriazine-based compounds (e.g., U.S.application No. 2006/0286646); reduction of substituted acetophenones(e.g., U.S. Pat. No. 6,800,477); and reduction of ketothiolanes (WO2005/054491).

Naturally occurring KRED enzymes can be found in a wide range ofbacteria and yeasts (for reviews: Kraus and Waldman, Enzyme catalysis inorganic synthesis Vols. 1&2. VCH Weinheim 1995; Faber, K.,Biotransformations in organic chemistry, 4th Ed. Springer, BerlinHeidelberg New York. 2000; Hummel and Kula Eur. J. Biochem. 1989184:1-13). Several KRED gene and enzyme sequences have been reported,including: Candida magnoliae (Genbank Acc. No. JC7338; GI:11360538);Candida parapsilosis (Genbank Acc. No. BAA24528.1; GI:2815409),Sporobolomyces salmonicolor (Genbank Ace. No. AF160799; GI:6539734);Lactobacillus kefir (Genbank Acc. No. AAP94029.1; GI: 33112056);Lactobacillus brevis (Genbank Acc. No. 1NXQA; GI: 30749782);Exiguobacterium acetylicum (Genbank Acc. No. BAD32703.1) andThermoanaerobium brockii (Genbank Acc. No. P14941; GI: 1771790).

The KRED catalyzed reduction of I-3 to I-4 requires that an electrondonor is present in the solution. Generally, a cofactor is used as theelectron donor in the KRED reduction reaction. The cofactor operates incombination with the KRED and/or GDH in the process. Suitable cofactorsinclude, but are not limited to, NADP⁺ (nicotinamide adeninedinucleotide phosphate), NADPH (the reduced form of NADP⁺), NAD⁺(nicotinamide adenine dinucleotide) and NADH (the reduced form of NAD⁺).Generally, the reduced form of the cofactor is added to the reactionmixture. Accordingly, the methods of the present disclosure are carriedout wherein an electron donor is present selected from NADPH cofactor orNADH cofactor. In certain embodiments, the method can be carried outwherein the reaction conditions comprise an NADH or NADPH cofactorconcentration of about 0.03-0.5 g/L, about 0.05-0.3 g/L, about 0.1-0.2g/L, about 0.5 g/L, about 0.1 g/L, or about 0.2 g/L.

In some embodiments of the process, a cofactor recycling system is usedto regenerate cofactor NADPH/NADH form NADP⁺/NAD⁺ produced in thereaction. A cofactor recycling system refers to a set of reactants thatreduce the oxidized form of the cofactor (e.g., NADP⁺ to NADPH) therebyallowing the KRED catalysis to continue. The cofactor recycling systemmay further comprise a secondary substrate and catalyst, for example,the substrate glucose, and the enzyme GDH, that catalyzes the reductionof the oxidized form of the cofactor by the reductant. Cofactorrecycling systems to regenerate NADH or NADPH from NAD⁺ or NADP⁺,respectively, are known in the art and may be used in the methodsdescribed herein. Suitable exemplary cofactor recycling systems that maybe employed include, but are not limited to, glucose and glucosedehydrogenase (GDH), formate and formate dehydrogenase (FDH),glucose-6-phosphate and glucose-6-phosphate dehydrogenase, a secondary(e.g., isopropanol) alcohol and secondary alcohol dehydrogenase,phosphite and phosphite dehydrogenase, molecular hydrogen andhydrogenase, and the like. These systems may be used in combination witheither NADP⁺/NADPH or NAD⁺ NADH as the cofactor. Electrochemicalregeneration using hydrogenase may also be used as a cofactorregeneration system. See, e.g., U.S. Pat. Nos. 5,538,867 and 6,495,023,both of which are incorporated herein by reference. Chemical cofactorregeneration systems comprising a metal catalyst and a reducing agent(for example, molecular hydrogen or formate) are also suitable. See,e.g., PCT publication WO 2000/053731, which is incorporated herein byreference.

In some embodiments of the present disclosure, the cofactor recyclingsystem can comprise glucose dehydrogenase (GDH), which is an NAD⁺ orNADP⁺-dependent enzyme that catalyzes the conversion of D-glucose(dextrose) and NAD⁺ or NADP⁺ to gluconic acid and NADH or NADPH,respectively. GDH enzymes suitable for use in the practice of theprocesses described herein include both naturally occurring GDHs, aswell as non-naturally occurring GDHs. Naturally occurring glucosedehydrogenase encoding genes have been reported in the literature, e.g.,the Bacillus subtilis 61297 GDH gene, B. cereus ATCC 14579 and B.megaterium. Non-naturally occurring GDHs generated using, for example,mutagenesis, directed evolution, and the like and are provided in PCTpublication WO 2005/018579, and US publication Nos. 2005/0095619 and2005/0153417.

In some embodiments, the co-factor recycling system can comprise aformate dehydrogenase (FDH), which is an NAD⁺ or NADP⁺-dependent enzymethat catalyzes the conversion of formate and NAD⁺ or NADP⁺ to carbondioxide and NADH or NADPH, respectively. FDHs suitable for use ascofactor regenerating systems in the KRED catalyzed reaction describedherein include naturally occurring and non-naturally occurring formatedehydrogenases. Suitable formate-dehydrogenases are described in PCTpublication WO 2005/018579. Formate may be provided in the form of asalt, typically an alkali or ammonium salt (for example, HCO₂Na,KHCO₂NH₄, and the like), in the form of formic acid, typically aqueousformic acid, or mixtures thereof. A base or buffer may be used toprovide the desired pH.

In some embodiments, the co-factor regenerating system can comprise thesame KRED enzyme that catalyzes the reduction of I-3 to I-4. In such anembodiment, the same KRED catalyzing the reduction of I-3 to I-4 alsocatalyzes the oxidation of a secondary alcohol (e.g., isopropanol toacetone oxidation) and thereby simultaneously reduces the NAD⁺ or NADP⁺to NADH or NADPH. Accordingly, in some embodiments, the KRED catalyzedconversion of I-3 to I-4 can be carried out in the presence of asecondary alcohol (e.g., IPA) and without any coenzyme (e.g., GDH)present in the solution for the recycling of the NADPH or NADH cofactor.In such embodiments, the suitable reaction conditions can comprise anIPA concentration is about 55-75% (v/v), an NADPH or NADH cofactorloading of about 0.03-0.5 g/L, and wherein no cofactor recycling enzymeis present other than the KRED itself.

Suitable cofactor recycling systems for use in the KRED catalyzedconversion of compound I-3 to I-4 include the co-enzyme glucosedehydrogenase (GDH) coupled with the substrate glucose (L- orD-glucose). Suitable GDH cofactors include, but are not limited to, theGDH of polypeptide of SEQ ID NO. 3 and GDH of polypeptide of SEQ ID NO.4, both of which are commercially available from Codexis Inc., RedwoodCity, Calif.

In one embodiment, compound I-5 is prepared by reacting compound I-4with an acetonide protection agent:

wherein R¹ is selected from the group consisting of C₁₋₆alkyl, benzyland phenyl; and P¹ is selected from the group consisting of Ac, Bn, Boc,Bz, Cbz, DMPM, FMOC, Ns, Moz, and Ts. In one embodiment, R¹ is selectedfrom the group consisting of methyl, ethyl, propyl, butyl and benzyl. Inanother embodiment, P¹ is Boc.

Suitable acetonide protection reagents include, but are not limited to,2,2-dimethoxy propane, 2,2-diethoxylpropane, 2-methoxypropene andacetone. In one embodiment, the acetonide protection reagent is2,2-dimethoxy propane.

The reaction from I-4 to I-5 can be carried out in the presence of asolvent. Suitable solvents include, but are not limited to, toluene,acetone, THF, IPAc, dichloromethane, EtOAc, MeCN, and mixtures thereof.In one embodiment, the solvent is a mixture of toluene and acetone.

The reaction from I-4 to I-5 can be carried out in the presence of anacid. Suitable acids include, but are not limited to, boron trihalides,organoboranes, HCl, H₂SO₄, TFA, H₃PO₄ and TiCl₄. In one embodiment, theacid is BF₃. In another embodiment, the BF₃ is in the form of borontrifluoride etherate (BF₃.O(Et)₂).

The reaction from I-4 to I-5 can be carried out at a temperature of 10°C. to 50° C., or more specifically, 20° C. to 40° C. It has been foundthat increasing the reaction temperature from 20° C. to 40° C. canincrease the rate of reaction by 2-3 fold while not affecting theproduct profile.

In one embodiment, compound I-6 is prepared from compound I-5 with areducing agent:

wherein R¹ is selected from the group consisting of C₁₋₆alkyl, benzyland phenyl; and P¹ is selected from the group consisting of Ac, Bn, Boc,Bz, Cbz, DMPM, FMOC, Ns, Moz, and Ts. In one embodiment, R¹ is selectedfrom the group consisting of methyl, ethyl, propyl, butyl and benzyl. Inanother embodiment, P¹ is Boc.

Suitable reducing agents include, but are not limited to, LiAlH₄, LiBH₄,NaBH₄—LiBr and DIBAL. In one embodiment, the reducing agent is LiAlH₄.In another embodiment, the reducing agent is LiBH₄. In yet anotherembodiment, the reducing agent LiBH₄ can be generated in situ, forexample by the use of a combination of NaBH₄ and LiBr.

The amount of the reducing agent is typically 0.8 to 1.6 equiv., or morespecifically, 1.0 to 1.4 equiv.

In one embodiment, the reduction from I-5 to I-6 is carried out at atemperature of 0° C. to 60° C., or more specifically, 0° C. to 35° C.,or even more specifically, 20° C. to 30° C. In one embodiment, thetemperature is 20° C. to 30° C.

In one embodiment, the oxidation of compound I-6 to compound I-7 iscarried out in the presence of a catalyst with an oxidizing agent:

wherein P¹ is selected from the group consisting of Ac, Bn, Boc, Bz,Cbz, DMPM, FMOC, Ns, Moz, and Ts. In one embodiment, P¹ is Boc.

Suitable oxidizing agents include, but are not limited to, NaOCl,NaClO₂, hydrogen peroxide, Swern oxidation and variants such as pyridinesulfur trioxide, PCC, and DCC. In one embodiment, the oxidizing agent isNaOCl.

The amount of the oxidizing agent is typically 1.1 equiv. to 1.3 equiv.,or more specifically, 1.2 equiv. to 1.25 equiv. In one embodiment, theamount of the oxidizing agent is 1.25 equiv.

Suitable catalysts for the above oxidation reaction include, but are notlimited to, TEMPO and TEMPO analogues. In one embodiment, the catalystis TEMPO.

One advantage of the presently described process is that compound I-7from the oxidation step can be used directly in the next Homer WadsworthEmmons (hereinafter, “HWE”) step to obtain compound I-8. This one potprocess eliminates the need for solvent switch and can increase theyield and reduce cost.

In one embodiment, the oxidation step from I-6 to I-7 can be carried outin the presence of a solvent. Suitable solvents include, but are notlimited to, THF, MTBE, CH₂Cl₂, MeCN, toluene and mixtures thereof. Inone embodiment, the solvent is a mixture of toluene and MeCN. In anotherembodiment, the solvent is a mixture of CH₂Cl₂ and MeCN.

In one embodiment, the HWE reaction between I-7 and A-4 is carried outin the presence of a solvent:

wherein P¹ and P² are each independently selected from the groupconsisting of Ac, Bn, Boc, Bz, Cbz, DMPM, FMOC, Ns, Moz, and Ts. In oneembodiment, both P¹ and P² are Boc.

Suitable solvents include, but are not limited to, THF, MTBE, CH₂Cl₂,MeCN, toluene and a mixture comprising two of the foregoing solvents. Inone embodiment, the solvent is the mixture of toluene and MeCN.

The HWE reaction is typically carried out at a temperature of −10 to 50°C., or more specifically, 0 to 40° C. In one embodiment, the temperatureis 0 to 25° C. In another embodiment, the temperature is 40° C.

The HWE reaction is typically carried out in the presence of a base or asalt. In one embodiment, the base is a tertiary amine. In anotherembodiment, the base is N,N-diisopropylethylamine (DIPEA).

In one embodiment, the salt is lithium halide, or more specifically,LiCl or LiBr.

In the HWE reaction, an impurity compound I-21 (aldol dimmer by-product)may be formed in addition to compound I-8:

It has been found that by adjusting pH to between 6.5 and 7.0 after thereaction, higher purity compound I-8 can be obtained with improvedyield. Additionally, addition of more reactant compound A-4 has beenshown to drive the impurity I-21 to product I-8. In one embodiment,addition of an extra 0.2 equiv. of A-4 can reduce the level of I-21 tofrom 8 LCAP to 2 LCAP.

Increasing the reaction temperature can speed up the conversion to thedesired product compound I-8 and reduce the level of the byproductcompound I-21.

By changing the reaction from a batch process to an addition controlledprocess, the yield of compound I-8 can be improved and the level ofbyproduct compound I-21 can be reduced. For example, by adding reactantcompound I-7 to a solution containing reactant compound A-4, the levelof I-21 can be decreased and the yield of compound I-8 improved.

In one embodiment, a solution containing 1.2 equiv of A-4, 3 equiv. ofDIPEA and 3 equiv. of LiCl in 5 volumes of MeCN was prepared and warmedto 40° C. A toluene stream of compound I-7 was then added to thismixture over 3 h, after an additional 30 min aging conversion to productwas complete. The level of impurity I-21 was about ˜1 LCAP. Sampling thereaction at 1 h intervals showed there was no build-up of compound I-7in the reaction mixture. After work up the product was isolated with a90% isolated yield.

It has also been found that using slightly smaller amount of reactantA-4 does not negatively affect the yield of compound I-8. In oneembodiment, 1.0 instead of 1.2 equiv. of compound A-4 was used and highyield was still obtained.

Compound A-4 used in the HWE reaction can be prepared from compound A-1:

using similar synthetic steps and conditions as described in A GeneralProcedure for the Preparation of β-Ketophosphonates, Maloney et. al., J.Org. Chem., 74, page 7574-7576 (2009).

In one embodiment, the reduction of compound I-8 to produce compound I-9is carried out in the presence of a catalyst:

wherein P¹ and P² are each independently selected from the groupconsisting of Ac, Bn, Boc, Bz, Cbz, DMPM, FMOC, Ns, Moz, and Ts. In oneembodiment, both P¹ and P² are Boc.

Suitable catalysts include, but are not limited to, Pd, Raney Ni, Pt,PdCl₂, and Pd(OH)₂. In one embodiment, the catalyst is 5% Pd/C.

In another embodiment, the reduction from I-8 to I-9 is carried out inthe presence of a solvent. Suitable solvents include, but are notlimited to, THF, MTBE, CH₂Cl₂, MeCN, toluene, methanol, ethanol,2-propanol and mixtures thereof. In one embodiment, the solvent is THF.

In another embodiment, the reduction reaction is carried out usinghydrogen gas at a pressure of 2 to 300 psig, preferably about 40 psig,in the presence of a catalyst.

In one embodiment, compound I-9 reacts with an acid to produce compoundI-10 through a cyclization reaction:

Suitable acids include, but are not limited to, HCl, HBr, TFA, MeSO₃H,TfOH, H₂SO₄, para-toluenesulfonic acid, and other sulfone acids such asRSO₃H wherein R is C₁₋₆alkyl, aryl or substituted aryl. In oneembodiment, the acid is HCl.

In one embodiment, HCl is used as acid and an HCl salt of compound I-10is obtained. In one embodiment, the HCl salt is in the form of bis-HClsalt. In another embodiment, the bis-HCl salt is in the form of amono-hydrate. In another embodiment, the mono-hydrate of the bis-HClsalt of compound I-10 is a crystalline material.

The conversion from I-9 to I-10 can be carried out at a temperature of 0to 40° C., or more specifically, 15 to 25° C., or even morespecifically, 20 to 25° C. In one embodiment, the temperature is 20 to25° C.

In one embodiment, compound I-10 is reduced to compound I-11 in thepresence of a catalyst:

The reaction conditions for the conversion from I-10 to I-11 can becontrolled so a cis-selective hydrogenation process is obtained. In oneembodiment, the cis-selective hydrogenation is carried out in thepresence of a catalyst. Suitable catalysts include, but are not limitedto Pt on alumina, Pd on alumina, Pd/C, Pd(OH)₂—C, Raney Ni, Rh/C, Rh/Al,Pt/C, Ru/C and PtO₂. In one embodiment, the catalyst is Pt on alumina.

In another embodiment, the cis-selective hydrogenation from I-10 to I-11is carried out in the presence of HMDS, which can protect the hydroxygroup in situ and therefore improve the diastereo selectivity. Othersuitable protecting reagents include, but are not limited to, TMSCl,TESCl, and TBDMSCl.

In one embodiment, compound I-11 is obtained in the form of acrystalline anhydrous free base. In another embodiment, compound I-11 isobtained in the form of a crystalline free base hemihydrate.

As used herein, the term “alkyl” means both branched- and straight-chainsaturated aliphatic hydrocarbon groups having the specified number ofcarbon atoms. For example, C₁₋₆alkyl includes, but is not limited to,methyl (Me), ethyl (Et), n-propyl (Pr), n-butyl (Bu), n-pentyl, n-hexyl,and the isomers thereof such as isopropyl (i-Pr), isobutyl secbutyl(s-Bu), tert-butyl (t-Bu), isopentyl, sec-pentyl, tert-pentyl andisohexyl.

As used herein, the term “aryl” refers to an aromatic carbocycle. Forexample, aryl includes, but is not limited to, phenyl and naphthale.

Throughout the application, the following terms have the indicatedmeanings unless noted otherwise:

Term Meaning Ac Acyl (CH₃C(O)—) Aq Aqueous Bn Benzyl BOC (Boc)t-Butyloxycarbonyl Boc₂O Di-tert-butyl dicarbonate Bz Benzoyl ° C.Degree celsius Calc. or calc'd Calculated Cbz Carbobenzyloxy CDI1,1′Carbonyldiimidazole DCC N,N′-Dicyclohexycarbodiimide DCMDichloromethane DKR Dynamic kinetic resolution DMAcN,N-dimethylacetamide DMAP 4-Dimethylaminopyridine DMFN,N-dimethylformamide DMPM 3,4-Dimethoxybenzyl DMSO Dimethyl sulfoxideEDC 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide Eq. or equiv.Equivalent(s) ES-MS and ESI-MS Electron spray ion-mass spectroscopy EtEthyl EtOAc Ethyl acetate FMOC 9-Fluorenylmethyloxycarbonyl g Gram(s) hor hr Hour(s) HATU O-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate HBTUO-(Benzotriazol-1-yl)-N,N,N′,N′- tetramethyluronium hexafluorophosphate)HCl Hydrogen chloride HMDS Hexamethyldisilazane HPLC High performanceliquid chromatography HOAc Acetic acid HOBT 1-Hydroxy-1H-benzotriazoleHOPO 2-Hydroxypyridine-N-oxide IPA Isopropyl alcohol kg Kilogram(s)LC/MS or LC-MASS Liquid chromatography mass spectrum L Liter(s) LAH orLiAlH₄ Lithium aluminium hydride LCAP Liquid Chromatography Area PercentLiBH₄ Lithium borohydride M Molar(s) Me Methyl MeCN Acetonitrile MeOHMethanol min Minute(s) mg Milligram(s) mL Milliliter(s) mmolMillimole(s) Moz or MeOZ p-Methoxybenzyl carbonyl MTBE Methyl tert-butylether NADP Nicotinamide adenine dinucleotide phosphate sodium salt nMNanomolar Ns 4-Nitrobenzene sulfonyl PCC Pyridinium chlorochromate 5%Pd/C Palladium, 5 weight percent on activated carbon Ph Phenyl r.t. orrt or RT RT Sat. Saturated TBDMSCl Tert-Butyldimethylsilyl chloride TEAor Et₃N Triethylamine TEMPO 1-Oxyl-2,2,6,6-tetramethylpiperidine TESClTriethylchlorosilane TFA Trifluoroacetic acid THF Tetrahydrofuran TMSClTrimethylchlorosilane Ts p-Toluene sulfonyl

Reaction Schemes below illustrate the synthetic steps, reagents andconditions employed in the synthesis of the compounds described herein.The synthesis of compound I-11 which is the subject of this inventionmay be accomplished by one or more of similar routes.

EXAMPLE 1 Preparation of Compound i-11 from Starting Compound i-1

In Scheme 1, one-pot through process produced keto amide i-3 fromstarting material i-1. An enzymatic DKR reduction produced i-4 from i-3.After protection, ester i-5 was reduced to alcohol i-6 with LAH orLiBH₄.

Once compound i-6 was obtained, it was converted to i-7 by TEMPOoxidation. For the TEMPO oxidation and subsequent HWE coupling step, aone-pot through process was used such that the crude stream of thealdehyde i-7 after phase cut was used directly for the HWE reaction toavoid solvent switch. Unsaturated ketone i-8 was isolated over 5 steps.Finally, compound i-8 was converted to compound i-11 through i-9 andi-10. Detailed experimental conditions are described below.

Step 1. Preparation of Compound i-3 from Compound i-1

To a mixture of Na₂CO₃ (110 g, 1.034 mol) in water (606 mL) and EtOAc(606 mL) at 0-5° C. was added glycine methyl ester (i-1, 119 g, 948mmol) in portions over 30 min. The resulting slurry was aged foradditional 15-30 min, PhCOCl (100 ml, 0.862 mol) was then added dropwiseover 1.5 h at 0-5° C. After aging additional 1 h at 0-5° C., thereaction mixture was warmed to 25° C. to form a homonogenous biphasicsolution. The separated organic phase was azetropically concentrated andsolvent switched to MeCN. A final volume of about 600 mL was obtained.

DMAP (43.1 mmol, 5.26 g) was added and then a solution of Boc₂O (0.948mol, 207 g) in MeCN (200 mL) was added at RT dropwise over 2-3 h. Thereaction solution was aged at RT for ˜6 h. The batch was vacuum degassedwith N₂ three times to remove CO₂ generated in the amidation step. THF(540 mL, KF<500) was added to the reaction solution. A solution oft-BuOK (1.12 mol, 128 g, 97%) in THF (670 ml) was then added at 0-10° C.dropwise over 1-2 h. The reaction solution was aged at 0-5° C. for 1 h.The reaction was then quenched with 15 wt % citric acid in water (0.431mol, 91 g citric acid in 515 mL H₂O) at <10° C. The organic phase waswashed with 480 mL of half saturated NaCl in water and solvent switchedto IPA to a final volume of ˜1.25 L with ˜10% water in IPA at <45° C.The batch was warmed to 40-50° C. Water (1250 mL) was added dropwise at40-50° C. over 2 h resulting in a slurry.

The slurry was cooled to ambient temperature (20° C.) and aged for 1-2 hbefore filtration. The wet cake of i-3 was displacement washed with 30%IPA in water (640 mL×2). Suction dry at RT or vacuum oven dry at <50° C.with dry N₂ sweep gave 227 g of white crystalline solid i-3 with 90%yield as determined by HPLC described below.

HPLC Method

Column: Asentis Express C18, 4.6×150 mm, 2.7 μm particle size;

Column Temp: 30° C.; Flow Rate: 1.5 mL/min; UV Detection: 210 nm;

Mobile Phase: A: 0.1% H₃PO₄ B: acetonitrile

Mobile Phase Program:

Time, min 0 1 5 7 9 A % 95 95 60 10 10 B % 5 5 40 90 90

Step 2. Preparation of Compound i-4 from Compound i-3 through DKRReduction

To a solution of K₂HPO₄ (14.1 g) in 80 mL water at RT was added dextrose(9.8 g) followed by NADP (360 mg), KRED enzyme of SEQ ID NO. 2 (290 mg)and a cofactor recycling system of SEQ ID NO. 4 (115 mg). The resultinghomogenous solution was pH adjusted to a minimum of 7.5 with 2M NaOHprior to use.

A solution of i-3 (12.0 g) in DMSO (36 ml) at 30° C. was added dropwiseto the above aqueous enzyme solution at 30° C. over 4 h under vigorousagitation. 2M NaOH (˜21 mL) was added dropwise to maintain the reactionmixture at pH of 7.3 to 7.7. Once 90% (˜19 mL) of 2M NaOH solution wasadded, the reaction temperature was raised to 35° C. until >95%conversion was achieved.

IPA (91 mL) and MTBE (49 mL) were added at RT. The organic layer wasseparated. The aqueous phase was extracted with IPA/MTBE (140 ml,IPA:MTBE=20:80). The combined organic phase was washed with brine (50mL, 10% w/v brine) and the crude product containing compound i-4 wasdirectly used for the next step.

Using the following HPLC method, the retention times of i-3 and i-4 wereabout 8.8 and 7.8 min, respectively.

HPLC Method

-   Column: Ace 3 Column C18, 3×150 mm, 3 μm particle size-   Column Temp: 30° C.; Flow Rate: 0.75 mL/min; UV Detection: 215 nm;-   Mobile Phase: A: Formate Buffer, pH 4 (1.26 g HCO₂Na and 0.79 mL    HCO₂H in 1 L H₂O)

B: Acetonitrile

-   Mobile Phase Program:

Time (min) % B 0 5 10 95Chiral SFC MethodColumn: Chiralpak IC, 250×4.6 mm, 5 μm particle sizeColumn Temp: 35° C.; Flow Rate: 2 mL/min; UV Detection: 210 nm;Mobile Phase Program:

Time, min 0 2 13 15 A % 95 95 60 60 MeOH % 5 5 40 40

Step 3. Preparation of Compound i-5 from Compound i-4 through AcetonideProtection

To a toluene solution of the i-4 ester (10.6 g, in ˜25 to 30 mL toluene,crude solution from previous DKR step) were added 50 mL of acetone and20 ml of 2,2-dimethoxy propane. A solution of BF₃ etherate (0.43 mL) intoluene (2 mL) was then added via a syringe pump at RT over 2 h. Thereaction solution was aged at RT for 15 h. The conversion was found tobe >97% by HPLC. The retention times of i-4 and i-5 were about 4.5 minand 6.5 min, respectively.

Et₃N (0.5 mL) was added dropwise. After aging for additional 15 min atambient temperature, the solution was solvent switched to toluene (˜30mL) while most of the acetone was removed in vacuum. MTBE (60 mL) wasadded and the organic phase was washed with 5% NaHCO₃/brine (40 mL).Organic phase was azetropically dried and solvent switched to toluene toa final volume of ˜35-40 mL.

HPLC Method

Column: Asentis Express C18, 4.6×150 mm, 2.7 μm particle size

Column Temp: 40° C.; Flow Rate: 1.5 mL/min; UV Detection: 210 nm;

Mobile Phase: A: 0.1% H₃PO₄ B: acetonitrile

Mobile Phase Program:

Time, min 0 5 7 10 A % 95 15 5 5 B % 5 85 95 95

Step 4. Preparation of Compound i-6 through Reduction of Compound i-5

Compound i-6 can be prepared from the reduction of compound i-5 undertwo alternative conditions.

Option 1: LAH Reduction:

To a crude i-5 ester solution in toluene (8.28 g assay, from previousprotection step, in ˜2.5-3 vol of toluene) was added 40 mL of THF. Then,the solution was cooled to 0° C. LiAlH₄ (2M solution in THF, 6.9 mL) wasadded dropwise over 1 h while the internal temperature was kept at 0-5°C. The reaction solution was aged for additional 0.5-1 h. Conversionwas >99% by HPLC. At 0-5° C., 0.52 mL of H₂O in 2.0 mL of THF followedby 0.52 mL of 15% NaOH then followed by 1.56 mL of H₂O were addedslowly. The slurry mixture was warmed up to RT and filtrated throughSolka Floc. The wet cake was washed with THF (25 mL) and toluene (16mL).

Assay yield of 91% was obtained using the following HPLC method. Theretention times of i-5 and i-6 were about 6.5 min and 5.7 min,respectively.

HPLC Method

Column: Asentis Express C18, 4.6×150 mm, 2.7 μm particle size;

Column Temp: 40° C.; Flow Rate: 1.5 mL/min; UV Detection: 210 nm;

Mobile Phase: A: 0.1% H₃PO₄ B: Acetonitrile

Mobile Phase Program:

Time, min 0 5 7 10 A % 95 15 5 5 B % 5 85 95 95Option 2: LiBH₄ Reduction

A crude solution of i-5 ester in toluene (11.1 g assay, from previousprotection step, in ˜2.5-3 vol of toluene) was added to a mixture ofLiBH₄ (0.937 g) in THF (60 mL) over 15-30 min. Then, the reactionsolution was aged for 15 h at 35° C. The conversion was >97% by HPLC.

The reaction solution was cooled down to RT and inversely quenched to asolution of 10% NH₄Cl (40 mL) while the internal temperature wasmaintained below 5° C. with external cooling. The quenched solution wasaged at ambient temperature for 2-3 h or until the evolution of H₂ gasceased. MTBE (100 mL) was added. Aqueous layer was discarded and organiclayer was solvent switched to toluene to a final volume of 40 mL, whichwas directly used in the subsequent oxidation step. Assay yield was 92%and aqueous loss was 1.2%.

Step 5. Preparation of Compound i-7 by TEMPO Oxidation of Compound i-6

To a solution of i-6 alcohol in toluene (20 g assay, ˜60 mL) was addedacetonitrile (120 mL) at RT. KBr (1.16 g), NaHCO₃ (1.8 g) and water (40mL) were then charged resulting in a biphasic mixture. The biphasicmixture was cooled to 5° C. and TEMPO (305 mg) was added. Then, NaClOsolution (Clorox; 6 wt %; 101 g) was added dropwise at 0-5° C. over 2 h.After addition, the reaction was stirred at 5° C. for ˜30 min.Conversion of >96% was obtained.

The reaction was quenched by dropwise addition of 10% sodium sulfite (50mL) at 5° C. The organic layer was separated and directly used for thesubsequent HWE coupling step without further purification. The assayyield was 17.5 g (88%) by ¹H NMR using DMAc as internal standard.

Retention times of i-6 and i-7 using the following HPLC method wereabout 3.3 min and 3.9 min, respectively.

HPLC Method

Column: Zorbax, Eclipse Plus C18, 4.6×50 mm, 1.8 μm particle size;

Column Temperature: 22° C.; Flow Rate: 1.5 mL/min; UV Detection: 210 nm;

Mobile Phase: A: 95/5/0.1, H₂O/Methanol/H₃PO₄ B: 95/5, MeCN/methanol

Mobile Phase Program:

Time, min 0 5 6 A % 60 10 10 B % 40 90 90

Step 6. Preparation of i-8 by Homer Wadsworth Emmons (HWE) CouplingReaction

To a solution of i-7 aldehyde in wet toluene/acetonitrile (162 gsolution; 17.5 g assay; 10.81 wt %) obtained above at ˜10° C. were addedacetonitrile (140 mL), phosphonate a-4 (24.6 g) and LiBr (14.9 g) whilethe internal temperature was maintained below 0° C.

The reaction was warmed up to 0° C., and Hunig's base (22.2 g) wascharged at 0-5° C. dropwise over 2 h. The resulting reaction mixture wasstirred at 0-5° C. for 2-4 h and allowed to warm to RT, followed byaging at RT for 12 h. HPLC showed conversion(product/(product+aldehyde)) of >99%.

The slurry was cooled to 5° C., and a 10% aqueous solution of citricacid (˜75 g) was added dropwise to adjust the pH to 6.5-7.0 whilemaintaining the batch temperature at 0-5° C. The aqueous phase wasseparated at 0-5° C. and discarded.

The organic layer was washed with saturated NaHCO₃ (57 mL) and with H₂O(57 mL) successively. The organic phase was solvent switched to IPA to afinal volume of ˜192 mL. The product was gradually crystallized duringthe distillation.

Water (16.4 mL, 0.6 vol.) was added, and the resulting slurry was heatedto 49° C. to give a homogeneous solution. The resulting solution wascooled to 40° C. and seeded (0.27 g). The resulting mixture was aged at40° C. for 2 h to establish a seed bed, and H₂O (93 mL) was chargeddropwise at 40° C. over 3 h, followed by aging at 40° C. for 1 h. Theslurry was allowed to cool to 5-10° C. over 2 h, followed by aging at5-10° C. for 2 h.

The wet cake was washed with 50% H₂O/IPA (a 164 mL cold displacementwash followed by a 110 mL slurry wash). Suction dried under nitrogengave the product as an off-white solid (24.9 g, 100 wt %, >99 LCAP, 80%isolated yield from aldehyde).

Using the following HPLC method, the retention times of i-7, a-4 and i-8were about 3.0 min, 1.2 min and 3.8 min, respectively.

HPLC Method

Column: Zorbax, Eclipse Plus C18, 4.6×50 mm, 1.8 μm particle size

Column Temp: 40° C.; Flow Rate: 1.5 mL/min; UV Detection: 210 nm;

Mobile Phase: A: 0.1% H₃PO₄ B: MeCN

Mobile Phase Program:

Time, min 0 3 7 A % 60 10 10 B % 40 90 90

Step 7. Preparation of Compound i-9 from Compound i-8

THF (84 g) followed by enone i-8 (19.07 g) and 10% Palladium on carbon(0.95 g) were charged to a hydrogenation vessel. The batch washydrogenated for 90 min at 25° C. until uptake of hydrogen had ceased.The catalyst was removed through filtration of a bed of solka floc. Thefiltered residues were washed with THF (84 g). The combined organicphase was solvent switched to IPA to a final volume of 142 mL, which wasdirectly used in the next step. Assay yield of 93% was obtained (17.8 gof i-9).

Using the following HPLC method, the retention times of i-8 and i-9 wereabout 11.2 min and 11.4 min, respectively.

HPLC Method

Column: HiChrom ACE C18 (250×4.6 mm), 3 μm particle size;

Column Temperature: 30° C.; Flow rate: 1.0 mL/min; Detection: 210 nm,254 nm;

Mobile phase: A: 1 mL of phosphoric acid (85%) dissolved in 1 L of H₂OB: MeCN

Mobile Phase Program:

Time, min 0 5 8 15 16 20 A % 95 65 5 5 95 95 B % 5 35 95 95 5 5

Step 8. Preparation of Compound i-10 from Compound i-9

To a solution of the N-Boc-Ketone aniline i-9 (26.1 assay kg) in IPA(˜125 g/L) was added 4N HCl in IPA (220.8 L) at RT. The reaction mixturewas stirred vigorously at 20-25° C. for 24 h. The batch was distilledunder reduced pressure, at constant volume by charging IPA up to onebatch volume, to remove HCl. The batch was then concentrated to a finalvolume of ˜215 L.

The resulting slurry was heated to 45° C., and IPAc (˜430 L) was slowlyadded to the batch over 2-3 h. The slurry was then cooled to ˜20° C.over 1-2 h and aged overnight. The batch was filtered, and the cake waswashed with a 1:2 mixture of IPA:IPAc (52 L) followed by IPAc (52 L).The wet cake was dried at 45° C. under nitrogen atmosphere to give thecyclic imine bis-HCl monohydrate salt i-10 (16.1 kg). The isolated yieldof 94% was obtained.

Using the same HPLC method as in Step 7 (i-8 to i-9), the retentiontimes of i-9 and i-10 (bis-HCl salt) were about 11.3 min and 8.3 min,respectively.

Step 9. Preparation of Compound i-11 from Compound i-10

To a mixture of imine dihydrochloride monohydrate i-10 (12.0 g, 98.5 wt%) in THF (86 mL) under N₂ was added hexamethyldisilazane (10.95 g)while maintaining the batch temperature below 25° C. The resultingslurry was stirred vigorously at ambient temperature for 2 h.

A 300 mL autoclave was charged with a suspension of 5% platinum onalumina (0.605 g) in THF (32 mL), followed by the substrate slurryprepared above. The resulting mixture was stirred at RT under hydrogen(40 psig) until the hydrogen uptake ceased. The completion of thehydrogenation was confirmed by HPLC, and the vessel was inerted withnitrogen.

The reaction mixture was discharged, and the vessel rinsed with 96 mL ofTHF. The batch was filtered through a pad of Solka Floc, and the pad wasrinsed with the THF vessel rinse (˜96 mL). The combined filtrate wasstirred with 0.5 M hydrochloric acid (129 mL) at ambient temperature for1 h. The aqueous layer was separated. IPAc (39 mL) followed by 5 Nsodium hydroxide (˜15 mL) was added to adjust the pH to 10.0 withvigorous stirring.

The organic layer (˜120 mL) was separated and treated with AquaGuardPowder (Meadwestvaco) (2.4 g) at RT for 2 h. The solution was filteredthrough a pad of Solka Floc, and the pad was rinsed with 2-propanol (18mL). The combined filtrate was concentrated to 70 mL. The solution wasdistilled at the constant volume by feeding a total of 140 mL of2-propanol, maintaining the batch temperature at 33-35° C. The resultingsolution was concentrated to ˜34 mL and heated to 50° C., followed byaddition of H₂O (6.3 mL). The resulting solution was cooled to 41-43° C.and seeded with pyrrolidine aniline hemihydrate (42 mg). The resultingmixture was aged at 41-43° C. for 1 h to establish a seed bed.

Water (60.9 mL) was charged at 41-43° C. over 6 h, and the resultingmixture was allowed to cool to 10° C. over 3 h, followed by aging at 10°C. for 2 h. The solids were collected by filtration and washed with 25%2-propanol/H₂O (50 mL). The wet cake was suction-dried at ambienttemperature under nitrogen to afford 7.68 g of pyrrolidine aniline i-11as hemihydrate.

¹H NMR (d₆-DMSO) δ 7.27 (m, 4H), 7.17 (m, 1H), 6.81 (d, J=8.1, 2H), 6.45(d, J=8.1 Hz, 2H), 5.07 (s, br, 1H), 4.75 (s, 2H), 4.18 (d, J=7.0 Hz,1H), 3.05 (m, 2H), 2.47 (dd, J=13.0, 6.7 Hz, 1H), 2.40 (dd, J=13.0, 6.6Hz, 1H), 1.53 (m, 1H), 1.34 (m, 1 HO, 1.22 (m, 2H).

¹³C NMR (d₆-DMSO) δ 146.5, 144.3, 129.2, 127.8, 127.4, 126.8, 126.7,114.0, 76.8, 64.4, 60.1, 42.1, 30.2, 27.2.

Using the following HPLC method, the retention times of i-10 (bis-HClsalt) and i-11 were about 8.3 min and 8.5 min, respectively.

HPLC Method

Column: Waters Xbridge C18, 150×4.6 mm, 3.5 μm;

Column Temperature: 25° C.; Flow rate: 1 mL/min; Detection: 210 nm, 254nm;

Mobile phase: A: Acetonitrile B: 0.1% aqueous NH₄OH adjusted to pH9.5with H

Mobile Phase Program:

Time, min 0 4 8 10 17 A % 99 65 65 30 30 B % 1 35 35 70 70

EXAMPLE 2 Preparation of Compound i-8 from Compound i-1—AlternativeConditions Step 1. Preparation of Compound i-3 from Compound i-1

To an inerted 1000 L vessel, water (178 kg) was charged, followed byglycine methyl ester hydrochloride (HCl salt of i-1, 35.0 kg) to form asolution. EtOAc (178 L; 160.6 kg) was charged and the mixture cooled to0° C. Triethylamine (56.4 kg; 78 L) was added over about 30 minmaintaining internal temperature <10° C. The mixture was cooled to 0° C.and benzoyl chloride (35.6 kg; 29.4 L) added over >30 min maintaininginternal temperature <10° C. HPLC analysis at the end of the benzoylchloride addition showed 100% conversion to benzoylated intermediate.

10% Phosphoric acid (prepared from 9 kg of 85 wt % phosophoric acid and81 kg H₂O) was then charged over 15 min and the mixture stirred. Thelower aqueous layer was removed and then back-extracted with EtOAc twice(178 L; 160.6 kg followed by 89 L; 80.3 kg). Loss to aqueous layer wasabout 2.1%.

The organic layers were combined and concentrated to ˜50 L, thenacetonitrile (178 L; 140.0 kg) charged and concentrated to ˜50 L.Acetonitrile (178 L; 140.0 kg) was then charged and the solution cooledto 0° C. and DMAP (3.09 kg) charged. Cooling to 0° C. was required toremain below the flash point of MeCN before charging solid via manway.

The mixture was warmed to 20° C. and a solution of Boc anhydride (60.8kg; 64.7 L) in MeCN (64.7 L; 60.8 kg+10 L rinse) was charged to themixture over 30 min. The reaction mixture was aged overnight. HPLCanalysis showed 100% conversion to Boc-intermediate.

The mixture was degassed via 3× vacuum/N₂ cycles, cooled to 0° C. and asolution of t-BuOK (36.9 kg) in THF (142 L; 126.6 kg) added over 1 hmaintaining internal temperature <5° C. The reaction mixture was agedfor 1 h at 5-10° C. HPLC analysis showed full conversion to keto-ester.

10% Citric acid (aq.) (prepared from 36.5 kg citric acid and 328.6 kgH₂O) was then added to the mixture, stirred and the lower aqueous layerremoved.

5% NaCl (aq.) (prepared from 14.25 kg NaCl and 270.8 kg H₂O) was addedto the organics, stirred and the lower aqueous layer removed. The lowerbrine layer was back-extracted with MTBE (80 kg) and all organicscombined.

The combined, washed organics were concentrated to ˜300 L, then IPA (178L; 139.7 kg) added and concentrated down to ˜285 L (1H-NMR showed noresidual MeCN) then IPA (117 kg) added. The slurry was heated to 50° C.to obtain a solution and H₂O (327.5 kg) added over 1 h maintaininginternal temperature at 40-50° C., then cooled to 20° C. over 45 min andaged overnight. Liquor loss was <2% by end of the cool-down.

The slurry was filtered and washed with IPA/H₂O (38.3 kg: 146.3 kg) andthe solids dried overnight at 50° C. in vacuo to give ketoester compoundi-3 as an off-white solid (65.25 kg; 99.4 LCAP; 100 LCWP) The yield was89%.

Using the HPLC method described below, the retention time of i-3 wasabout 4.0 min.

HPLC Method

Column: Ascentis Express C18, 100×4.6 mm, 2.7 μm particle size;

Column Temperature: 40° C.; Flow rate: 1.8 mL/min; Detection: 210 nm,220 nm, 254 nm;

Mobile phase: A: 1.0 ml of 99.9% phosphoric acid (85 w/w %) in 1 L ofH₂O B: MeCN

Gradient:

Time, min 0 6 8 9 10 A % 90 5 5 90 90 B % 10 95 95 10 10

Step 2. Preparation of Compound i-4 from Compound i-3 through DKRReduction

A 0.1M phosphate buffer solution was prepared by dissolving sodiumphosphate dibasic dihydrate (8.54 kg) in water (480 kg). The pH wasadjusted to 7.0 using HCl (approx 1.5 L).

0.1M phosphate buffer (400 kg) was charged to a 1000 L vessel followedby DMSO (66 kg). D-(+)-glucose (39.2 kg) and NADP disodium salt (0.90kg) was charged via the manway and the mixture stirred at 30° C. untilall solids had dissolved.

The remaining 0.1M phosphate buffer (88.5 kg) was charged to a 400 Lvessel, followed by the KRED enzyme of SEQ ID NO. 1 (3.0 kg) and acofactor recycling system of SEQ ID NO. 3 (0.30 kg). This mixture wasstirred slowly at 20° C. until all enzymes had dissolved. Violentstirring should be avoided to prevent form from forming in the mixture.Once dissolved, a hazy yellow solution was obtained.

To a 160 L vessel was charged the keto ester substrate i-3 (32.6 kg)followed by DMSO (66 kg). The mixture was stirred at 30° C. until allthe solid had dissolved.

The pH of the glucose/NADP solution was adjusted from 7.19 to 7.50 using2M sodium hydroxide solution (2.4 kg).

The enzyme solution was then transferred to the 1000 L vessel, followedby a water line wash (5 kg). The pH of the combined solution was thenre-adjusted from 7.30 to 7.50 by the addition of 2M sodium hydroxidesolution (1.9 kg).

The DMSO solution of the keto ester was then charged to the 1000 Lvessel over approximately 4 h, maintaining the temperature in thereaction vessel at 30° C. and maintaining the pH between 7.3 and 7.7.

Starting material crystallized during addition. Reaction mixture wasthen a slurry throughout. The pH became more acidic as the reactionprogressed. The range of 7.3 to 7.7 was maintained by the addition of 2MNaOH.

The reaction was then aged at 30° C., with the pH maintained between 7.3and 7.7 until reaction was complete. Reaction achieved 90% completionafter approximately 5 days. Total uptake of 2M NaOH was 55 kg. Projectedtotal uptake was 60 kg.

The extraction workup was performed in two halves due to vessel volumelimitations. The amounts detailed below are for the total batch size.

MTBE (900 L) and methanol (400 L) were charged and the mixture stirredand allowed to settle. A two phase mixture was obtained—upper clearorganic layer containing the product and a lower hazy aqueous layercontaining all of the enzyme residues. Separation was good but didrequire a 30-60 min settling time.

The phases were separated and the aqueous extracted with MTBE (300 L).The organic phases were combined and washed with 10% brine (150 L).Total losses to aqueous layers were 1-2% of theory yield.

The organic layer was then concentrated from 1100 L to 95 L bydistillation at reduced pressure. Toluene (185 kg) was charged and thebatch concentrated to a final volume of 100 L. Batch temperaturemaintained below 40° C. during distillation.

Total material processed was 64.2 kg. Assay yield was 53.9 kg (86%).Contained ˜14% keto ester i-3. ee>99.9%. KF<25 ppm. MTBE not detected by¹H NMR.

Using the HPLC method described below, the retention times of i-3 andi-4 were about 4.5 min and 3.8 min, respectively.

HPLC Method

Column: Ascentis Express C18, 100×4.6 mm, 2.7 μm particle size;

Column Temperature: 40° C.; Flow rate: 1.8 mL/min; Detection: 210 nm;

Mobile phase: A: 1.0 ml of 99.9% phosphoric acid (85 w/w %) in 1 L ofH₂O B: MeCN

Gradient:

Time, min 0 6 8 A % 90 5 5 B % 10 95 95

Step 3. Preparation of Compound i-5 from Compound i-4 through AcetonideProtection

To an inerted 1000 L vessel, the hydroxyester i-4 in toluene (53.9 kghydroxyester; total solution 114.1 kg) was charged, followed by acetone(257 L; 203 kg) and 2,2-dimethoxypropane (97 L; 82 kg) and the mixturestirred for 5 min. Boron trifluoride diethyl etherate (2.20 L; 2.47 kg)was charged to the mixture over 30 min and aged 15 h overnight at 20° C.(no exotherm was noted). Reaction time was ˜4 h for completion; HPLCanalysis after overnight showed >99% conversion.

Triethylamine (2.4 L; 1.76 kg) was then charged and the mixtureconcentrated to low volume (˜100 L). MTBE (308 L; 229 kg) was charged,followed by 5% NaHCO₃ (aq.) (129 L prepared from 6.45 kg NaHCO₃ and122.55 kg H₂O) and 10% NaCl (aq.) (129 L prepared from 12.9 kg NaCl116.1 kg H₂O). The mixture was stirred for 5 min, the layers allowed tosettle and the lower aqueous layer removed. The washed organics wereconcentrated to ˜75 L, then toluene charged (91 L; 105 kg) andconcentrated to ˜75 L. Toluene (138 L; 119 kg) was then added. Reactiontime was ˜4 h for completion; HPLC analysis after overnight showed >99%conversion.

Triethylamine [2.4 L; 1.76 kg] was then charged and the mixtureconcentrated to low volume, ˜100 L. MtBE [308 L; 229 kg] was charged,followed by 5% NaHCO₃ (aq.) [129 L prepared from 6.45 kg NaHCO₃ and122.55 kg DI H₂O] and 10% NaCl (aq.) [129 L prepared from 12.9 kg NaCl116.1 kg DI H₂O]. The mixture was stirred for 5 min., the layers allowedto settle and the lower aqueous layer removed. The washed organics wereconcentrated to ˜75 L, then toluene charged [91 L; 105 kg] andconcentrated to ˜75 L. Toluene [138 L; 119 kg] was then added.

Final solution was 176.8 kg and product acetonide i-5 assay was 61.2 kg(100%). Keto ester compound I-3 was still present at 14.2 LCAP.

Using the same HPLC method as in Step 1 of this Example, the retentiontimes of i-4 and i-5 were about 3.2 min and 5.2 min, respectively.

Step 4. Preparation of Compound i-6 through Borohydride Reduction ofCompound i-5

To a 1000 L vessel was charged tetrahydrofuran (218 kg) and 10% lithiumborohydride solution in THF (50.8 kg). The mixture was stirred and thetemperature adjusted to 20° C.

A solution of acetonide ester i-5 (containing ˜14 LCAP keto ester i-3carried through from the enzymatic DKR step) in toluene (55.9 kg ofsubstrate in a total of 176.8 kg of toluene solution) was charged over45 min maintaining the batch temperature between 20 and 25° C. The batchwas warmed to 35° C. and aged for 18 h. Small exotherm was noted duringcharging of substrate. Minimal off-gassing was observed. HPLC showed <1%starting material remaining.

The batch was transferred to a 400 L vessel followed by a THF line wash(10 kg). To the 1000 L vessel was charged a solution of ammoniumchloride (24.5 kg) in water (245 kg). The ammonium chloride solution wascooled to 0-5° C. and then the batch charged over 90 min, maintainingthe temperature between 0 and 5° C. Hydrogen gas evolved. Rate ofaddition of batch to ammonium chloride quench solution was controlled soas not to pressurise vessel.

The quenched reaction mixture was warmed to 20° C. and stirred for 2 h(until off-gassing ceased). Agitation was stopped and the layers allowedto settle. The lower aqueous phase was run out and the organic washedwith ˜10% brine (5 kg sodium chloride in 50 kg water). There was someemulsion at interface and so the majority of the organics were removedand the emulsion and aqueous layer re-extracted with toluene (100 kg).The organic phases were combined and concentrated at reduced pressure to100 L (batch temperature maintained below 40° C.).

Heptane (410 kg) was charged and the mixture warmed to 30° C. The batchwas washed with a mixture of acetonitrile (20 kg) in water (300 kg) for20 min. The aqueous layer was removed and the wash repeated twice more.Washing performed at 30-35° C. to maintain solubility. Diol compoundi-16 which has the following structure was reduced from 14 to 2 LCAP:

The organic phase was then concentrated to 100 L under reduced pressure.Acetonitrile (120 kg) was charged and the batch concentrated to 140 L(142 kg). Assay yield was 55.6 kg (99%) and <2 LCAP of i-16 was present.

Using the same HPLC method as in Step 2 of this Example, the retentiontimes of i-5, i-6 and i-16 diastereomers were about 5.6 min, 4.9 min,3.0 min and 3.1 min respectively.

Step 5. Preparation of Aldehyde Compound i-7 by TEMPO Oxidation ofCompound i-6

To an inerted 1000 L glass-lined vessel were charged KBr (2.65 kg),NaHCO₃ (4.11 kg) and water (152 kg). The toluene/CH₃CN/heptane solutionof the alcohol i-6 (142 kg at 39.2 wt %) was added to the aqueoussolution, followed by acetonitrile (83 kg) and toluene (153 kg). Theresulting stirred solution was cooled to 0-2° C. Using a diaphragm pump,a solution of TEMPO (0.695 kg) in toluene (2.8 L) was added to thereaction mixture, rinsing the line and pump with toluene (˜1 L).

Using an air-driven Teflon-lined pump, sodium hypochlorite (120 kg, 99L, 13.9 wt % active chlorine) was charged to the vessel through anabove-surface addition line. The sodium hypochlorite solution was thenadded to the reaction mixture over a period of 70 min while maintainingthe batch temperature below 10° C. The addition-line was rinsed into thebatch with water (1 kg). The batch was aged at <5° C. for 20 min. Thesodium hypochlorite does not need to be pre-cooled to 15° C., but shouldnot be at temperatures above ambient (i.e. 23° C.). The addition rateshould be maintained within 60-70 min. A longer addition rate will incurlarger amounts of both starting material and over-oxidized acidby-product, whilst a shorter run time will probably not be possible dueto exothermic activity. The exotherm will cease as soon as addition ofthe bleach stops. In this case, 4.0 LCAP starting material, 1.3 LCAPacid and 87.4 LCAP aldehyde was obtained. A further 0.05 eq. NaOCl (4kg) was charged before proceeding with the quench, although the batchwas not re-assayed again.

The temperature of the batch was checked at 5° C., and 1 M sodiumsulfite (total prepared from 5.04 kg of Na₂SO₃ and 35 kg water; actualamount added was only 18 L of this solution) was then added over aperiod of 10 min, maintaining the batch temperature below 10° C. Thebatch temperature was set to 20° C. and aged for 5 min. The batch wastested with starch iodide paper to ensure no oxidant was present.

The bi-phasic mixture was mixed for 10 min and then allowed to settleout. The lower aqueous layer was removed and disposed of. The toporganic cut was washed with water (68 kg). The lower aqueous cut asagain removed and disposed of. The organic layer was again tested foroxidant with starch iodide paper. (Good hold point-hold solution at 5°C.).

Both cuts were very good and settled fairly quickly. The final organiccut indicated 1.3 LCAP starting material and 91.1 LCAP aldehyde. In bothaqueous layers, residual KBr and the acid by-product were removed.

The organic solution was then distilled, under vacuum, to a final volumeof −135 L whilst maintaining temperature <40° C. (˜3 volumes based onyield of aldehyde). KF should be <2% water for the subsequent HWE step.In this case the KF was 80 μg/mL.

This afforded a 36.8 wt % solution of aldehyde (131.7 kg total; 36.8 wt%, 48.5 kg assay for i-7; 87% yield (¹H-NMR assay with anisole asinternal standard), which was stored in a plastic lined steel drum undernitrogen at 5° C. whilst awaiting further processing. Using the HPLCmethod described below, the retention times of i-6 and i-7 were about4.9 min and 5.5 min, respectively.

HPLC method

Column: Ascentis Express C18, 100×4.6 mm, 2.7 μm particle size;

Column Temperature: 40° C.; Flow rate: 1.5 mL/min; Detection: 210 nm,254 nm;

Mobile phase: A: 1.0 ml of 99.9% phosphoric acid (85 w/w %) in 1 L ofH₂O B: MeCN

Mobile Phase Gradient:

Time, min 0 5 7.5 7.51 A % 90 10 10 90 B % 10 90 90 10

Step 6. Preparation of i-8 by HWE Coupling of i-7 Aldehyde with Compounda-4

The phosphonate a-4 (67.4 kg, 1.2 equiv.) and lithium chloride (19.98kg, 3 equiv.) were charged to a 1000 L vessel, followed by acetonitrile(188.64 kg). The mixture was cooled to ˜5° C. andN,N-Diisopropylethylamine (60.95 kg, 3 equiv.) was added whilemaintaining an internal temperature below 20° C. Once addition wascomplete the batch was warmed to 40° C. and the solution of aldehyde i-7in toluene from the previous step was added over 3 h then aged for afurther 30 min.

Adding the aldehyde solution to the prepared ylide resulted in a verylow level (˜1 LCAP) of aldol dimmer by-product I-21 at the end of thereaction:

The batch was cooled to 0° C. and MTBE (177.6 kg) was added followed bycitric acid (10% aq.) to adjust the pH to between 6.5 and 7 (6.87achieved in the plant). If the pH was overshot then addition of 2N NaOHwas conveniently used to adjust the pH back into range.

The aqueous lower layer was run off and the organics were washed with10% sodium bicarbonate solution (240 L), then water (2×120 L). Duringthe first aqueous wash there was some solid in the aqueous layeridentified as citric acid salts precipitated due to super saturation.This was conveniently ran off with the aqueous layer.

An assay yield of the final solution showed there was 75.67 kg, 90.1% inthe organic stream (Note: this was an estimate based on approximatevolume in the vessel) Losses to the aqueous cuts were <0.1%.

The solution was distilled to a minimum volume (˜100 L) and iso-propylalcohol (604.5 kg) was added. The solution was then distilled to a finalvolume of 616 L (˜7 volumes+ product), and the batch was heated to 49°C. Water (46.2 kg, 0.6 volumes) was added and the batch was cooled to40° C. over 30 min. The batch was seeded and allowed to age for 1 h at40° C. to establish a seed bed. Water (492.8 kg) was then added to thebatch over 1 h whilst marinating the batch temperature at 40° C. Thebatch was aged for 2 h at 40° C. then cooled to 10° C. over 2 h, andaged at this temperature over night. After the age period an assay ofthe liquors showed there was a liquor concentration of 2.9 mg/ml. Thebatch was filtered and the cake was washed with 1:1 of IPA:water (380 L)which had been cooled to 10° C. The solid was dried in vacuo at 40° C.Then co-milled to afford 75.93 kg of the desired product, 100 LCAP, 99wt %, 89% yield. Losses to the liquors and washes were 3%.

With the success of this protocol the reaction was attempted using only1.01 equiv of phosphonates a-4, this resulted in a 99% assay yield atthe end of reaction.

Using the same HPLC method as in Step 2 of this Example, the retentiontimes of a-4 and i-8 were about 3.8 min and 6.2 min, respectively.

Using procedures similar to Steps 7-9 of Example 1, compound i-11 can beprepared from compound i-8.

While the invention has been described and illustrated with reference tocertain particular embodiments thereof, those skilled in the art willappreciate that various changes, modifications and substitutions can bemade therein without departing from the spirit and scope of theinvention. It is intended, therefore, that the invention be defined bythe scope of the claims which follow and that such claims be interpretedas broadly as is reasonable.

What is claimed is:
 1. A process for producing compound I-11:

comprising: (a) reacting compound I-4:

with an acetonide protection reagent selected from the group consistingof 2,2-dimethoxy propane, 2,2-diethoxylpropane, 2-methoxypropene andacetone, to produce compound I-5:

(b) reducing said compound I-5 with a reducing agent at a temperature of0° C. to 40° C. to produce compound I-6;

(c) oxidizing said compound I-6 with an oxidizing agent in the presenceof a solvent and a catalyst to produce compound I-7:

(d) reacting said compound I-7 with phosphonate compound A-4:

to produce compound I-8:

(e) reducing said compound I-8 in the presence of a catalyst to producecompound I-9;

(f) reacting said compound I-9 with an acid to produce compound I-10:

and (g) reducing said compound I-10 in the presence of a catalyst toproduce compound I-11; wherein said P¹ and said P² are eachindependently selected from the group consisting of acyl (Ac), benzyl(Bn), t-butyloxycarbonyl (Boc), benzoyl (Bz), carbobenzyloxy (Cbz),3,4-dimethoxybenzyl (DMPM), 9-fluorenylmethyloxycarbonyl (FMOC),4-nitrobenzene sulfonyl (Ns), p-methoxybenzyl carbonyl (Moz), andp-toluene sulfonyl (Ts); and said R¹ is selected from the groupconsisting of C₁₋₆alkyl, benzyl, and phenyl.
 2. The process of claim 1,wherein in step (c): said solvent is selected from the group consistingof tetrahydrofuran (THF), methyl tert-butyl ether (MTBE),dichloromethane (CH₂Cl₂), acetonitrile (MeCN), toluene and a mixturecomprising two of the said foregoing solvents; said oxidizing agent isselected from the group consisting of sodium hypochlorite (NaOCl),sodium chlorite (NaClO₂), hydrogen peroxide, pyridine sulfur trioxide,pyridinium chlorochromate (PCC), and N,N′-dicyclohexycarbodiimide (DCC);and said catalyst is 1-oxyl-2,2,6,6-tetramethylpiperidine (TEMPO) or aTEMPO analogue.
 3. The process of claim 1, wherein said reaction betweensaid compound I-7 and said compound A-4 in step (d) is carried out at atemperature of 20 to 40° C. and in the presence of a solvent selectedfrom the group consisting of THF, MTBE, CH₂Cl₂, MeCN, toluene and amixture comprising two of said foregoing solvents.
 4. The process ofclaim 1, wherein said catalyst in step (e) is selected from the groupconsisting of Pd, Raney Ni, Pt, PdCl₂, and Pd(OH)₂; and said reducing iscarried out in the presence of hydrogen gas.
 5. The process of claim 1,wherein said acid in step (f) is selected from the group consisting ofHCl, HBr, trifluoroacetic acid (TFA), MeSO₃H, TfOH, H₂SO₄,para-toluenesulfonic acid, and RSO₃H, wherein R is an alkyl, an aryl, ora substituted aryl.
 6. The process of claim 1, wherein said reducing instep (g) is carried out in the presence of hexamethyldisilazane (HMDS)and said catalyst is selected from the group consisting of Pt onalumina, Pd on alumina, Pd/C, Pd(OH₂)—C, Raney Ni, Rh/C, Rh/Al, Pt/C,Ru/C, and PtO₂.
 7. The process of claim 1, further comprising a step ofreducing compound I-3:

in the presence of a ketoreductase (KRED) enzyme to produce compoundI-4:


8. The process of claim 7, wherein said KRED enzyme comprises an aminoacid sequence selected from the group consisting of the amino acidsequence of SEQ ID NOEL 1 and the amino acid sequence of SEQ ID NO: 2.9. The process of claim 8, further comprising a cofactor recyclingsystem comprising an amino acid sequence selected from the groupconsisting of the amino acid sequence of SEQ ID NO: 3 and the amino acidsequence of SEQ ID NO:
 4. 10. The process of claim 9, further comprisinga cofactor selected from the group consisting of NADH and NADPH.
 11. Theprocess of claim 7, further comprising a step of reacting compound I-1:

with benzoyl chloride and a protecting reagent to produce compound I-3:


12. A process for producing compound I-11:

comprising: (a) reacting compound I-1:

with benzoyl chloride and a protecting reagent to produce compound I-3:

(b) reducing said compound I-3 in the presence of a ketoreductase (KRED)enzyme to produce compound I-4:

(c) reacting said compound I-4 with an acetonide protection reagentselected from the group consisting of 2,2-dimethoxy propane,2,2-diethoxylpropane, 2-methoxypropene and acetone, to produce compoundI-5:

(d) reducing said compound I-5 with a reducing agent at a temperature of0° C. to 40° C. to produce compound I-6;

(e) oxidizing said compound I-6 with an oxidizing agent in the presenceof a solvent and a catalyst to produce compound I-7:

(f) reacting said compound I-7 with phosphonate compound A-4:

to produce compound I-8:

(g) reducing said compound I-8 in the presence of a catalyst to producecompound I-9;

(h) reacting said compound I-9 with an acid to produce compound I-10:

and (i) reducing said compound I-10 in the presence of a catalyst toproduce compound I-11; wherein said P¹ and said P² are eachindependently selected from the group consisting of acyl (Ac), benzyl(Bn), t-butyloxycarbonyl (Boc), benzoyl (Bz), carbobenzyloxy (Cbz),3,4-dimethoxybenzyl (DMPM), 9-fluorenylmethyloxycarbonyl (FMOC),4-nitrobenzene sulfonyl (Ns), p-methoxybenzyl carbonyl (Moz), andp-toluene sulfonyl (Ts); and said R¹ is selected from the groupconsisting of C₁₋₆alkyl, benzyl, and phenyl.
 13. The process of claim12, wherein said KRED enzyme in step (b) comprises an amino acidsequence selected from the group consisting of the amino acid sequenceof SEQ ID NO: 1 and the amino acid sequence of SEQ ID NO:
 2. 14. Theprocess of claim 13, wherein step (b) further comprises a cofactorrecycling system comprising an amino acid sequence selected from thegroup consisting of the amino acid sequence of SEQ ID NO: 3 and theamino acid sequence of SEQ ID NO:
 4. 15. The process of claim 14,further comprising a cofactor selected from the group consisting of NADHand NADPH.
 16. The process of claim 1, wherein R¹ is C₁₋₆alkyl.
 17. Theprocess of claim 7, wherein R¹ is C₁₋₆alkyl.
 18. The process of claim11, wherein R¹ is C₁₋₆alkyl.
 19. The process of claim 12, wherein R¹ isC₁₋₆alkyl.